Marijuana is the most widely used illicit drug in the United States. Delta-9-tetrahydrocannabinol (THC), the major psychoactive component in marijuana, has been reported to be immunosuppressive and to alter the functional activities of macrophages. Thus, definition of the effects of cannabinoids on macrophage function is important in view of initiatives to use marijuana as a medicinal for treatment of chronic disabling diseases, including MDS. It has been proposed that cannabinoid effects on cellular functions are exerted by both receptor and nonreceptor-mediated modes. We have shown that THC alters macrophage processing of hen egg lysozyme (HEL) and have implicated the peripheral cannabinoid receptor CB2 in this process. We have demonstrated also that THC and the potent cannabinoid analog CP55940 alter the inducible expression of select macrophage cytokine mRNAs, especially that of IL6. In addition, we have shown that THC inhibits the post-translational cleavage of the presecretory form of TNFalpha. In this proposed continuation study we will establish whether a functional linkage exists between a cannabinoid receptor and THC-mediated altered expression of the macrophage cytokines IL-1alpha, IL 1beta, IL6, and TNFalpha. Functional assessment of a role of a cannabinoid receptor in mediating cytokine expression will be achieved through experiments employing pertussis toxin uncoupling, beta-t2cAMP reconstitution, the paired stereospecific cannabinoid enantiomers HU210/HU211 and 1-Nantradol/d-Nantradol for definition of structural activity relationships, the CB1 and CB2 receptor selective antagonists SR141716A and SR144528, CB1 and CB2 receptor specific anti-sense oligonucleotides, and CB1 and CB2-specific antibodies. Functional linkage studies will be followed by those to define the site of action. Electromobility shift and supershift assays to determine effects on activation of the transcriptional factor NfkappaB, nuclear run-on experiments, promoter activity assay, and mRNA stability experiments will be performed to provide insight as to the step at which cannabinoids alter IL6 mRNA levels. Radiolabel biosynthetic incorporation pulse and pulse-chase experiments, Western immunoblotting, and immunofluorescence studies using cytokine-specific antibodies will be performed to establish whether expression of cytokines is affected at the translational, post- translational, and/or compartmentation levels. Finally, because we have demonstrated that THC inhibits the processing of HEL, we will examine potential sites of action at which this process is effected. Three major aspects of antigen processing will be examined, including disruption of disulfide bonds in an antigen, enzymatic activity of acid proteases called cathepsins and the status of the intracellular reducing environment.